Translation blocking morpholino

One- to two-cell stage embryos were injected with 1 to 2 nl of 0.3 mM translation-blocking morpholino (GeneTools) targeting irf8 (irf8 MO^atg; 5′-TCAGTCTGCGACCGCCCGAGTTCAT-3′) [57 (link)]. Off-target effects were controlled by comparison to uninjected embryos and embryos injected with Random Control-25N morpholino mixture (GeneTools) injected at concentration equal to the experimental morpholino. Morpholinos were prepared as a 1-mM stock solution, which was diluted to working concentration and coinjected with 0.05% phenol red solution. MO-injected embryos included for analysis were morphologically normal and survived at rates comparable to embryos injected with random control morpholino (59% to 74% for Random Control-25N, 74% for irf8 MO^atg).

myo1Cb-pCS2+, myo1D-pCS2+, and myo1D^tj16b-pCS2+ were linearized with KpnI and RNA in vitro synthesized using the mMessage mMachine SP6 transcription kit (Ambion). The resulting RNAs were injected at 50 ng/µl for myo1Cb and 25 ng/µl for myo1D and myo1D^tj16b. Arl13b-GFP-pCS2+ was linearized with ApaI and transcribed with SP6 RNA polymerase as previously described^{28 (link)}. RNA was injected at 20 ng/µl. To inhibit myo1D function, a translation-blocking morpholino (Gene Tools) with the sequence 5′-ACTTTCGTGTTCTGCCATAATAAGC-3′ was injected at 750 µM. All reagents were injected at the one cell stage at a volume of 0.88 nl together with 0.2% phenol red.

Translation-blocking morpholino (5′-GCTCGGTTTCCATCATGTTATACAT-3′) against the ATG-containing sequence was synthesized by Gene Tools. Morpholino was diluted to 0.3 mM with RNase-free water and injected into one-cell stage embryos and then raised in E3 medium at 28.5°C for imaging.
The Sox2 cDNAs containing the open reading frame were cloned into PCS2⁺ vector. After this recombinant plasmid was linearized, the Sox2 mRNA was synthesized in vitro by the mMESSAGE mMACHIN Kit (Ambion, USA) according to the manufacturer’s instruction, and then the capped mRNAs were purified using RNeasy Mini Kit (Qiagen, Hilden, Germany). Two nanoliters of Sox2 mRNA was injected at 20 ng/μl into one-cell stage embryos.
The Sox2 ORF was cloned from the zebrafish by PCR, and then cloned into a pME-MCS vector to produce middle entry clone (pME-Sox2). p5E-mnx1 plasmid was obtained from Addgene. To generate an expression construct, p5E-mnx1, pME-Sox2, and p3E-polyA were combined with pDestTol2pA2 by the LR recombination reaction as described in the Lifetech Multiste Gateway Manual (Figure 5E; Life Technologies, Carlsbad, CA, USA). Subsequently, this construct was co-injected with tol2-transposase mRNA into one-cell stage Sox2 mutant zebrafish embryos to create the mosaic rescue zebrafish model.